The overall objective of the proposed research is to identify effective and safe chemopreventive agents that will facilitate the development of cancer-preventive strategies and their application in the clinical setting. The antiestrogen Tamoxifen (Tam), which is used in the adjuvant therapy of breast cancer and is being evaluated as a prophylactic agent in women at high risk of breast cancer, increases the risk of endometrial cancer. Long-term estrogen replacement therapy for postmenopausal syndrome also increases endometrial cancer risk. Metabolism of Tam and estrogen occurs in the liver with subsequent accumulation of the metabolites in target tissues. 4-OHTam, a major metabolite of Tam, accumulates in human uterus. 17B-Estradiol (E2), a natural estrogen, is hydroxylated at the 4-position in human uterine cells by cytochrome P450 (CYP) isozyme lB1. Expression of CYPs in human uterine endometrium suggests that endometrial tissue has potential to generate genotoxic Tam metabolites. Peroxidase-mediated oxidation of 4-OHTam and 4-OHE2 to quinone derivatives is believed to play a role in DNA ariduct formation by these metabolites. The redox cycling between 4-OHTam and its phenoxy radical and quinone hydroquinone forms of estrogen may also contribute to genotoxicity by inducing oxidative DNA damage. If unrepaired, DNA modification has potential to initiate carcinogenesis. Human endometrium is rich in peroxidase activity. Other enzyme systems potentially involved in metabolism of 4-OHTam by one-electron oxidation pathway are also present in this tissue. This proposal hypothesizes that inhibition of peroxidase-driven activation of 4-OHTam and 4-OHE2 by antioxidants will reduce the genotoxic potential of Tam and/or estrogen administration in endometrial tissue. The specific aims are; a) to expose a human endometrial explant culture model to 4-hydroxy metabolites of Tam and E2 and detect the formation of DNA adducts, depurinating adducts and 8-hydroxy deoxyguanosine as indicators of DNA damage, b) to asses the chemopreventive efficacy of antioxidants ascorbate (vitamin C) and N-acetylcysteine (NAC) to inhibit DNA damage, and c) to investigate whether Tam alters the expression of CYPs in this model and thus alters tissue-specific Tam metabolism and subsequent DNA damage. The long-term goal of this proposal is to evaluate if the combination of Tam or estrogen with nontoxic antioxidants will cooperate to reduce uterine cancer risk associated with chronic exposure to Tam or estrogen.